U.S. patent application number 14/238908 was filed with the patent office on 2014-07-31 for protein synthesis kit, and method for expressing and extracting proteins using automatic extraction equipment.
This patent application is currently assigned to BIONEER CORPORATION. The applicant listed for this patent is You Sang Cho, Ji Won Han, Jun Ho Jung, Nam Il Kim, Han Oh Park. Invention is credited to You Sang Cho, Ji Won Han, Jun Ho Jung, Nam Il Kim, Han Oh Park.
Application Number | 20140212919 14/238908 |
Document ID | / |
Family ID | 48175583 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140212919 |
Kind Code |
A1 |
Park; Han Oh ; et
al. |
July 31, 2014 |
PROTEIN SYNTHESIS KIT, AND METHOD FOR EXPRESSING AND EXTRACTING
PROTEINS USING AUTOMATIC EXTRACTION EQUIPMENT
Abstract
Provided is a method of protein synthesis. The method of protein
synthesis according to the present invention uses an automatic
biological material purification apparatus including: a well plate
kit; a heating part; and a magnetic field applying part, such that
a plurality of target proteins may be more quickly and simply
obtained as compared to target proteins obtained by using the
existing method for expressing/purifying proteins through
conventional cell culture, and a reproducible synthesis efficiency
on the same proteins may be obtained due to no deviation between
reaction wells.
Inventors: |
Park; Han Oh; (Daejeon,
KR) ; Cho; You Sang; (Daejeon, KR) ; Jung; Jun
Ho; (Seoul, KR) ; Han; Ji Won; (Daejeon,
KR) ; Kim; Nam Il; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Park; Han Oh
Cho; You Sang
Jung; Jun Ho
Han; Ji Won
Kim; Nam Il |
Daejeon
Daejeon
Seoul
Daejeon
Daejeon |
|
KR
KR
KR
KR
KR |
|
|
Assignee: |
BIONEER CORPORATION
Daejeon
KR
|
Family ID: |
48175583 |
Appl. No.: |
14/238908 |
Filed: |
August 23, 2012 |
PCT Filed: |
August 23, 2012 |
PCT NO: |
PCT/KR2012/006715 |
371 Date: |
February 14, 2014 |
Current U.S.
Class: |
435/68.1 |
Current CPC
Class: |
B01J 2219/00364
20130101; B01J 19/0046 20130101; C12P 21/00 20130101; C07K 1/22
20130101; B01J 2219/00459 20130101; B01J 2219/00495 20130101; B01J
2219/00315 20130101; C12P 21/02 20130101 |
Class at
Publication: |
435/68.1 |
International
Class: |
C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 26, 2011 |
KR |
10-2011-0085824 |
Aug 17, 2012 |
KR |
10-2012-0090149 |
Claims
1. A method of protein synthesis, the method comprising: (1)
preparing a template for cell-free protein synthesis; (2) adding
the template to a cell-free protein expression solution to express
proteins; (3) adding magnetic particles coupled with an affinity
tag to the expressed protein to attach a target protein to the
magnetic particles; and (4) separating the attached target protein
from the magnetic particles, wherein expression and purification of
the target protein are simultaneously performed in an automatic
system using an automatic biological material purification
apparatus including: a heating part; and a magnetic field applying
part.
2. The method of claim 1, wherein a second multi well plate kit
420' and a first multi well plate kit 420 are used, the second
multi well plate kit 420' including: (a) a solution for diluting
the template for cell-free protein synthesis; (b) the cell-free
protein expression solution for expressing the target protein from
the template; and the first multi well plate kit 420 including: (c)
a magnetic particle solution for attaching the target protein to
the magnetic particles; (d) an eluting solution for the target
protein; (e) a magnetic particle reaction solution for coupling the
target protein with the magnetic particle and a washing
solution.
3. The method of claim 1, wherein the template for cell-free
protein synthesis is deoxyribonucleic acid (DNA) having a circular
form or a linear form.
4. The method of claim 3, wherein the DNA having a linear form is
produced by obtaining a primary reactant by amplifying a target
gene in a sample using a prepared primer set so as to amplify the
target gene and provide overlapping sequences at 5' and 3'
terminals; and secondarily amplifying the obtained primary reactant
using a premix for PCR containing the following composition A,
[Composition A] (a) an upstream cassette set and a downstream
cassette set positioned at both 5' and 3' terminals of the target
gene, and (b) a secondary primer set having overlapping sequences
at both 5' and 3' terminals of the cassette set.
5. The method of claim 4, wherein the composition A contains 0.1 to
0.5 ng/ul of cassette set for encoding an affinity tag at both 5'
and 3' terminals of the target gene and 0.1 to 1.0 pmoles/ul of
each of secondary forward and reverse primers having overlapping
sequences at 5' and 3' terminals of the cassette set.
6. The method of claim 4, wherein the affinity tag is
histidine.
7. The method of claim 4, wherein the magnetic particle contains
metal ions.
8. The method of claim 7, wherein the metal ion is an nickel
ion.
9. The method of claim 1, comprising: Injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; First mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; Second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
Mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; Heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
Preparing step S70 of a magnetic particle reaction mixture; Protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; Reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; Removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; Washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; Removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; Target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and Target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
10. The method of claim 9, wherein the washing step S150 washing
impurities except for the target protein and the removing step S170
removing the mixture except for the magnetic particles having the
target proteins attached thereto are sequentially performed once or
more times.
11. The method of claim 9, wherein the target protein containing
solution obtaining step S200 includes injecting the target protein
containing solution into a protein storage tube 442-3.
12. The method of claim 9, wherein the magnetic particle is a
magnetic particle coupled with an nickel ion.
13. The method of claim 2, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
14. The method of claim 3, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
15. The method of claim 4, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
16. The method of claim 5, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
17. The method of claim 6, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
18. The method of claim 7, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
19. The method of claim 8, comprising: injecting step S10 injecting
the template for cell-free protein synthesis into a unit well of
the multi well plate kit 420'; first mixing step S20 mixing a DEPC
distilled water injected into the unit well of the multi well plate
kit 420' in order to dilute the template with the injected
template; second mixing step S30 mixing a cell-free protein
expression solution with the mixture of the first mixing step;
mixture preparing step S40 preparing a protein synthesis reaction
solution by mixing the mixture of the second mixing step with a
cell disrupted liquid; heating step S50 applying heat to the
protein synthesis reaction solution in the specific unit well by
heating a lower portion of the specific unit well of the multi well
plate kit 420' having the mixture using a heating part 720;
preparing step S70 of a magnetic particle reaction mixture; protein
expression injecting step S110 injecting the protein expression
injected into the unit well of the multi well plate kit 420' into
the prepared magnetic particle reaction mixture; reacting step S120
reacting the protein expression injected into the unit well of the
multi well plate kit 420' with the magnetic particle; removing step
S140 removing a mixture except for the magnetic particles and
proteins coupled with the magnetic particle by applying a magnetic
field to the mixture containing the protein expression; washing
step S150 washing impurities except for the target protein from the
magnetic particle by injecting a washing solution injected into the
unit well of the multi well plate kit 420; removing step S170
removing a mixture except for the magnetic particles having the
target proteins attached thereto from a washing solution containing
mixture by applying a magnetic field to the washing solution
containing mixture; target protein separating step S180 separating
the target protein by injecting the eluting solution for the target
protein injected into the unit well of the multi well plate kit 420
into a mixture obtained from the removing step; and target protein
containing solution obtaining step S200 obtaining the target
protein containing solution except for the magnetic particles from
the eluting solution for the target protein containing the target
protein separated from the magnetic particles by applying a
magnetic field to the mixture.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for producing
proteins, and more particularly, to a method for producing proteins
including simultaneously performing expression and purification of
target proteins in an automatic system using an automatic
biological material purification apparatus including: a heating
part; and a magnetic field applying part.
BACKGROUND ART
[0002] As a method for expressing recombinant proteins, a method
for mainly using cells such as Escherichia coli and yeast,
transforming a recombinant protein expression vector into the cell,
and culturing the transformed cells to express proteins has been
generally used. The above-described method requires a strain
selection process which stably expresses recombinant protein and
has a difficulty in expressing protein having toxicity in a cell,
such that it takes at least several days to months to obtain one
protein.
[0003] Recently, a cell-free protein expression method synthesizing
protein in a test tube without using cells has received attention,
and various products related thereto has been developed. The
cell-free protein expression method, which is a system of
expressing protein by adding a template DNA capable of expressing
protein, for example, an expression vector, a PCR product, a cell
lysate, an expression solution, and a diethylpyrocarbonate (DEPC)
distilled water into a tube, and performing a reaction at a proper
temperature (30 to 40.degree. C.) for a proper time (1 to 3 hours),
has an advantage in that proteins having toxicity in a cell are
capable of being expressed, and a required time may be remarkably
reduced as compared to the above-mentioned method for expressing
proteins using cells. Here, in order to express proteins, proper
temperature and time are required. The reason is that an activity
on an enzyme should be accompanied during all processes including
synthesis of RNA from DNA in a tube and synthesis of protein from
the RNA, wherein the above-described processes are capable of being
performed in the case of maintaining a temperature when activities
of a plurality of enzymes are shown, that is, 30 to 40.degree.
C.
[0004] Since a plurality of proteins are mixed in a sample having
expressed recombinant proteins, in order to easily purify the
recombinant protein expressed from the sample, the expressed
proteins should have an affinity with a specific material.
Recently, a method for purification proteins by expressing a
coupled state between recombinant protein and histidine and using
an affinity of histidine and metal ions (nickel ion, cobalt ion,
and the like) has been mainly used, and various products related
thereto have been on sale. Among the methods, a method for using
magnetic particles has been mainly used, wherein metal ions on a
surface of the magnetic particles are coupled with histidine of the
target protein, such that the target protein only is capable of
being extracted from a plurality of proteins.
[0005] However, according to the existing technologies according to
the related art, high yield and high-purity purification may not be
achieved and it is difficult to control protein complicated
expression due to cell culture, which is non-effective, such that a
basic issue which is reproducible production on the same proteins
still has not been overcome.
[0006] Therefore, a method for producing proteins capable of
achieving high yield and high-purity purification and having
reproducible synthesis efficiency on the same protein is
required.
Technical Problem
[0007] The present inventors found a method for synthesis proteins
including simultaneously performing expression and extraction of
target proteins to be more effectively and easily performed as
compared to the related art in a continuous study of an expression
system effectively and easily controlling an expression of proteins
and a method for biosynthesizing proteins using the same, thereby
completing the present invention.
[0008] An object of the present invention is to provide a method
for synthesis proteins including simultaneously performing
expression and extraction of target proteins in an automatic system
using an automatic biological material purification apparatus
including: a heating part; and a magnetic field applying part.
[0009] In addition, an object of the present invention is to
provide a protein synthesis kit simultaneously performing
expression and purification of target proteins by combining a
method for expressing cell-free protein with a method for
purification protein using magnetic particles coupled with an
affinity tag.
Technical Solution
[0010] In order to achieve objects of the present invention, there
is provided a method for producing proteins capable of synthesizing
and producing up to 16 kinds of target proteins within 6 hours
using an automatic biological material purification apparatus
including: a heating part; and a magnetic field applying part and
applied by a combination of a method for expressing a cell-free
protein with a method for purification proteins using magnetic
particles coupled with an affinity tag.
[0011] Hereinafter, the present invention will be described in
detail.
[0012] In one general aspect, a method for producing proteins
includes:
[0013] (1) preparing a template for cell-free protein
synthesis;
[0014] (2) adding the template to a cell-free protein expression
solution to express proteins;
[0015] (3) adding magnetic particles coupled with an affinity tag
to the expressed protein to attach a target protein to the magnetic
particles; and
[0016] (4) separating the attached target protein from the magnetic
particles,
[0017] wherein expression and purification of the target protein
are simultaneously performed in an automatic system using an
automatic biological material purification apparatus including: a
heating part; and a magnetic field applying part.
[0018] A second multi well plate kit 420' and a first multi well
plate kit 420 may be used, the second multi well plate kit 420'
including:
[0019] (a) a solution for diluting the template for cell-free
protein synthesis;
[0020] (b) the cell-free protein expression solution for expressing
the target protein from the template; and
[0021] the first multi well plate kit 420 including: (c) a magnetic
particle solution for attaching the target protein to the magnetic
particles;
[0022] (d) an eluting solution for the target protein;
[0023] (e) a magnetic particle reaction solution for coupling the
target protein with the magnetic particle and a washing
solution.
[0024] In another general aspect, a method for producing proteins
includes:
[0025] (1) preparing a template for cell-free protein
synthesis;
[0026] (2) adding the template to a cell-free protein expression
solution to express proteins;
[0027] (3) adding magnetic particles coupled with an affinity tag
to the expressed protein to attach a target protein to the magnetic
particles; and
[0028] (4) separating the attached target protein from the magnetic
particles,
[0029] wherein an automatic biological material purification
apparatus including: a heating part; and a magnetic field applying
part is used,
[0030] a second multi well plate kit 420' and a first multi well
plate kit 420 are used, the second multi well plate kit including:
(a) a solution for diluting the template for cell-free protein
synthesis; (b) the cell-free protein expression solution for
expressing the target protein from the template; and the first
multi well plate kit including: (c) a magnetic particle solution
for attaching the target protein; (d) an eluting solution for the
target protein; (e) a magnetic particle reaction solution for
coupling the target protein with the magnetic particle and a
washing solution, and expression and purification of the target
protein are simultaneously performed in an automatic system.
[0031] The template for cell-free protein synthesis may be
deoxyribonucleic acid (DNA) having a circular form or a linear
form. Preferably, a plasmid DNA may be used as the DNA having a
circular form, a PCR product may be used as the DNA having a linear
form, but the present invention is not limited thereto.
[0032] In detail, the DNA having a linear form may be produced by
obtaining a primary reactant by amplifying a target gene in a
sample using a prepared primer set so as to amplify the target gene
and provide overlapping sequences at 5' and 3' terminals and a
premix for PCR; and
[0033] secondarily amplifying the obtained primary reactant using
the premix for PCR (hereinafter, referred to as `a kit for PCR`)
containing the following composition A,
[0034] [Composition A]
[0035] (a) an upstream cassette set and a downstream cassette set
positioned at both 5' and 3' terminals of the target gene, and
[0036] (b) a secondary primer set having overlapping sequences at
both 5' and 3' terminals of the cassette set.
[0037] The premix for PCR, which is a mixture containing mixed
components required for an amplification reaction, may contain
deoxynucleotide triphosphate mixture (dNTP mixture) and a buffer,
and thermostable DNA polymerase such as Taq DNA polymerase. In
addition, the premix for PCR may be prepared in a liquid or dried
form.
[0038] In the present invention, a kit for PCR used in producing
DNA having a linear form may contain a premix for PCR, 0.1 to 0.5
ng/ul of cassette set for encoding an affinity tag at both 5' and
3' terminals of a target gene and 0.1 to 1.0 pmoles/ul of each of
secondary forward and reverse primers having overlapping sequences
at 5' and 3' terminals of the cassette set.
[0039] In the present invention, a term: `cassette` is collectively
referred to as oligonucleotide having double strand which is
shortly synthesized so as to be artificially connected to DNA
product. Since a phosphate group is not found at 5' terminal of the
cassette, a single strand only in cassette consisting of double
strands may be connected to a genome DNA.
[0040] In the present invention, a term: `primer`, which is a singe
strand nucleic acid sequence having a short and free 3' hydroxyl
group, indicates a short nucleic acid sequence forming a template
and a base pair of a complementary nucleic acid and functioning as
a starting point for a strand copy of an nucleic acid template. The
primer may initiate DNA synthesis in the presence of and reagent
for polymerization reaction (that is, DNA polymerase) and different
four dNTPs under appropriate buffer and temperature. A kit for PCR
of the present invention includes a specific primer to a nucleotide
sequence of a target gene and a specific primer to a cassette
sequence.
[0041] In the present invention, the automatic biological material
purification apparatus including: a heating part; and a magnetic
field applying part may include a pipette block 100 having a
plurality of separable pipettes 141 and 142 mounted thereon and
pipetting a biological sample containing a target material to each
of the plurality of pipettes 141 and 142; a fixing body 200
supporting the pipette block 100; a pipette block up-and-down
moving part mounted on the fixing body 200 and vertically moving
the pipette block 100; a pipette block back-and-forth moving part
horizontally moving the fixing body 200 to horizontally move the
pipette block 100; a base plate 400 positioned in a lower portion
of the fixing body 200 and mounted with multi well plate kits 420'
and 420 having a plurality of unit wells configuring two column
wells adjacent to each other; a magnetic field applying part 700
positioned in a lower portion of a specific unit well of the multi
well plate kits 420' and 420 in order to apply a magnetic field to
the specific unit well of the multi well plate kits 420' and 420
and remove the magnetic field therefrom; and heating part 810
heating the specific unit well of the multi well plate kits 420'
and 420.
[0042] In addition, in the automatic biological material
purification apparatus including: a heating part; and a magnetic
field applying part according to the present invention, the
magnetic mounting part having a magnet mounted thereon and a
heating part for heating are vertically moved, such that
temperature may be controlled while applying or removing a magnetic
field, and well inserting grooves which are a plurality of columns
formed in the magnet mounting part cover the lower portion of the
unit well of the multi well plate kits, such that a reaction
efficiency may be more increased.
[0043] In detail, the magnetic field applying part 700 may include
a magnet mounting part 710 on which a magnet 711 is mounted; and a
lifting part 760 lifting up and down the magnet mounting part 710;
wherein the magnet mounting part 710 has unit well inserting
grooves 713 formed thereon so as to insert the lower portion of the
specific unit well of the multi well plate kits 420' and 420, and
the base plate 400 has unit well exposing holes 400-3 so that at
the time of lifting up the magnet mounting part 710, the lower
portion of the specific unit well of the multi well plate kits 420'
and 420 inserts the unit well inserting groove 713, the lower
portion of the specific unit cell of the multi well plate kits 420'
and 420 is spaced apart by a predetermined distance from the lower
portion of the unit well of the multi well plate kits 420' and 420
adjacent thereto so as to insert the unit well inserting groove
713, and the magnet 711 is inserted and mounted around the unit
well inserting groove 713.
[0044] The method may include: Injecting step S10 injecting the
template for cell-free protein synthesis into a unit well of the
multi well plate kit 420';
[0045] First mixing step S20 mixing a DEPC distilled water injected
into the unit well of the multi well plate kit 420' in order to
dilute the template with the injected template;
[0046] Second mixing step S30 mixing a cell-free protein expression
solution with a mixture of the first mixing step;
[0047] Mixture preparing step S40 preparing a protein synthesis
reaction solution by mixing the mixture of the second mixing step
with a cell disrupted liquid;
[0048] Heating step S50 applying heat to the protein synthesis
reaction solution in the specific unit well by heating a lower
portion of the specific unit well of the multi well plate kit 420'
having the mixture using a heating unit 720;
[0049] Preparing step S70 of a magnetic particle reaction
mixture;
[0050] protein expression injecting step S110 injecting the protein
expression injected into the unit well of the multi well plate kit
420' into the prepared magnetic particle reaction mixture;
[0051] Reacting step S120 reacting the protein expression injected
into the unit well of the multi well plate kit 420' with the
magnetic particle;
[0052] Removing step S140 removing a mixture except for the
magnetic particles and proteins coupled with the magnetic particle
by applying a magnetic field to the mixture containing the protein
expression;
[0053] Washing step S150 washing impurities except for the target
protein from the magnetic particle by injecting a washing solution
injected into the unit well of the multi well plate kit 420;
[0054] Removing step S170 removing a mixture except for the
magnetic particles having the target proteins attached thereto from
a washing solution containing mixture containing the washing
solution by applying a magnetic field to the mixture containing the
washing solution containing mixture;
[0055] Target protein separating step S180 separating the target
protein by injecting the eluting solution for the target protein
injected into the unit well of the multi well plate kit 420 into a
mixture obtained from the removing step; and
[0056] Target protein containing solution obtaining step S200
obtaining the target protein containing solution except for the
magnetic particles from the eluting solution for the target protein
containing the target protein separated from the magnetic particles
by applying a magnetic field to the mixture.
[0057] In detail, the injecting step S10, which injects the
template for cell-free protein synthesis into a unit well L of the
multi well plate kit 420' using the plurality of pipettes 141 and
142 mounted on the automatic biological material purification
apparatus including the heating part and the magnetic field
applying part, is performed and after the injecting step S10, a
first mixing step S20 is performed.
[0058] The first mixing step S20, which mixes a DEPC distilled
water H for diluting the template for the cell-free protein
synthesis injected into the unit well of the multi well plate kit
420' with the template for the cell-free protein synthesis injected
to the unit well L of the multi well plate kit 420' using the
plurality of pipettes 141 and 142 mounted in the automatic
biological material purification apparatus including the heating
part and the magnetic field applying part, is performed and after
the first mixing step S20, a second mixing step S30 is
performed.
[0059] The second mixing step S30, which mixes a cell-free protein
expression solution injected with the specific unit well G of the
multi well plate kit 420' with the unit well L injected with the
template for the cell-free protein synthesis mixed with the DEPC
distilled water injected into the unit well of the multi well plate
kit 420' using the pipettes 141 and 142, is performed and after the
second mixing step S30, a mixture preparing step S40 is
performed.
[0060] The mixture preparing step S40, which prepares a protein
synthesis reaction solution by mixing a cell disrupted liquid
injected into a cell disrupted liquid storage tube 442-1 with the
unit well L injected with the template for the cell-free protein
synthesis mixed with the DEPC distilled water injected into the
unit well of the multi well plate kit 420' using the pipettes 141
and 142, is performed and after the mixture preparing step S40, a
heating step S50 is performed.
[0061] The heating step S50, which is a first heating step applying
heat to the protein synthesis reaction solution in the specific
unit well L by heating a lower portion of the specific unit well L
of the multi well plate kit 420' using the heating part 720 in a
state in which the lower portion of the specific unit well L of the
multi well plate kit 420' inserts a unit well inserting groove 713
by lifting up the magnet mounting part 710, is performed and after
the heating step S50, a first protein expression injecting step S60
is performed.
[0062] The first protein expression injecting step S60, which
injects the first protein expression reacted in the protein
synthesis reaction solution in the specific unit well L into a unit
well J of the multi well plate kit 420' using the pipettes 141 and
142, is performed and after the first protein expression injecting
step S60, a preparing step S70 of a magnetic particle reaction
mixture is performed.
[0063] The preparing step of the magnetic particle reaction mixture
S70, which mixes the magnetic particle reaction solution injected
into a unit well A of the multi well plate kit 420 with the
magnetic particles injected into a unit well F of the multi well
plate kit 420 using the pipettes 141 and 142, is performed and
after the preparing step of the magnetic particle reaction mixture
S70, an injecting step of the reaction solution mixed with the
magnetic particle S80, a first magnetic field applying step S90,
and a first removing step S100 may be further performed in
sequence.
[0064] The injecting step of the reaction solution mixed with the
magnetic particles S80, which is a second injecting step injecting
the mixture mixed with the magnetic particle reaction solution into
the specific unit well L of the multi well plate kit 420' using the
pipettes 141 and 142 in a state in which the magnet mounting part
710 is lifted down, is performed, and after the second injecting
step S80, the first magnetic field applying step S90 is
performed.
[0065] The first magnetic field applying step S90, which applies a
magnetic field to the lower portion of the specific unit well of
the multi well plate kit 420' from the magnet mounting part 710 by
lifting up the magnet mounting part 710 so as to insert the lower
portion of the specific unit well L of the multi well plate kit
420' into the unit well inserting groove 713, is performed, and
after the first magnetic field applying step S90, a first removing
step S100 is performed.
[0066] The first removing step S100, which removes a mixture except
for the magnetic particles and materials attached to the magnetic
particle from the mixture mixed with the magnetic particle reaction
solution using the pipettes 141 and 142 in a state in which the
magnetic particles and the materials attached to the magnetic
particle in the mixture mixed with the magnetic particle reaction
solution are attached to an inner wall of the lower portion of the
specific unit well of the multi well plate kit 420' by a magnetic
field applied to the lower portion of the specific unit well L of
the multi well plate kit 420', is performed, and after the first
removing step S100, a protein expression injecting step S110 is
performed.
[0067] The protein expression injecting step S110, which injects
the first protein expression injected into the unit well J of the
multi well plate kit 420' into the unit well L of the multi well
plate kit 420' using the pipettes 141 and 142, is performed, and
after the second protein expression injecting step S110, a reacting
step S120 is performed.
[0068] The reacting step S120, which reacts the second protein
expression injected into the unit well L of the multi well plate
kit 420' with the magnetic particle injected into the unit well L
of the multi well plate kit 420' using the pipettes 141 and 142, is
performed, and after the reacting step S120, a second magnetic
field applying step S130 may be further performed.
[0069] The further performed second magnetic field applying step
S130, which applies a magnetic field to the lower portion of the
specific unit well L of the multi well plate kit 420' from the
magnet mounting part 710 by lifting up the magnet mounting part 710
so as to insert the lower portion of the specific unit well L of
the multi well plate kit 420' into the unit well inserting groove
713, is performed, and after the second magnetic field applying
step S130, a removing step S140 is performed.
[0070] The removing step S140, which is a second removing step
removing a mixture except for the magnetic particles and proteins
coupled with the magnetic particles using the pipettes 141 and 142
in a state in which the magnetic particles having proteins attached
thereto in the mixture mixed with the second protein expression are
attached to an inner wall of the lower portion of the specific unit
well L of the multi well plate kit 420' by a magnetic field applied
to the lower portion of the specific unit well L of the multi well
plate kit 420', is performed, and after the second removing step
S140, a washing step S150 is performed.
[0071] The washing step S150, which separates impurities except for
the target protein from the magnetic particle by injecting a
washing solution injected into the unit well of the multi well
plate kit 420 into the specific unit well L of the multi well plate
kit 420' using the pipettes 141 and 142 in a state in which the
magnet mounting part 710 is lifted down, is performed, and after
the washing step S150, a removing step S170 is performed.
[0072] The removing step S170, which is a third removing step
removing a mixture except for the magnetic particles having the
target proteins attached thereto in the mixture mixed with the
washing solution using the pipettes 141 and 142 in a state in which
the magnetic particles having the target proteins attached thereto
in the mixture mixed with the washing solution are attached to an
inner wall of the lower portion of the specific unit well of the
multi well plate kit 420' by a magnetic field applied to the lower
portion of the specific unit well of the multi well plate kit 420',
is performed, and after the removing step S170, a target protein
separating step S180 is performed.
[0073] The washing step S150 washing impurities except for the
target protein and the removing step S170 removing the mixture
except for the magnetic particles having the target proteins
attached thereto may be sequentially performed once or more times,
and after the washing step 150, a third magnetic field applying
step S160 may be further performed.
[0074] The further performed third magnetic field applying step
S160 applies a magnetic field to the lower portion of the specific
unit well L of the multi well plate kit 420' from the magnet
mounting part 710 by lifting up the magnet mounting part 710 so as
to insert the lower portion of the specific unit well L of the
multi well plate kit 420' into the unit well inserting groove
713.
[0075] The target protein separating step S180 performed after the
removing step S170 separates the target protein by injecting a
target protein eluting solution injected into the unit well of the
multi well plate kit 420 into the specific unit well L of the multi
well plate kit 420' using the pipettes 141 and 142 in a state in
which the magnet mounting part 710 is lifted down, and after the
target protein separating step S180, a fourth magnetic field
applying step S190 may be further performed.
[0076] The further performed fourth magnetic field applying step
S190 applies a magnetic field to the lower portion of the specific
unit well of the multi well plate kit 420' from the magnet mounting
part 710 by lifting up the magnet mounting part 710 so as to insert
the lower portion of the specific unit well L of the multi well
plate kit 420' into the unit well inserting groove 713 and after
the fourth magnetic field applying step S190, a target protein
containing solution obtaining step S200 is performed.
[0077] The target protein containing solution obtaining step S200
may obtain the target protein containing solution which is a
mixture except for the magnetic particles in the protein eluting
solution containing the target protein separated from the magnetic
particle using the pipettes 141 and 142 in a state in which the
magnetic particles in the target protein eluting solution
containing the target protein separated from the magnetic particle
are attached to an inner wall of the lower portion of the specific
unit well L of the multi well plate kit 420' by a magnetic field
applied to the lower portion of the specific unit well L of the
multi well plate kit 420' to produce the target protein.
[0078] In the present invention, the target protein containing
solution obtaining step S200 may include injecting the target
protein containing solution into a protein storage tube 442-3
mounted on the base plate 400 using the pipettes 141 and 142.
[0079] In the present invention, the magnetic particle may be a
magnetic particle coupled with an affinity tag of the target
protein, and more particularly, may include a metal ion,
preferably, may be a magnetic particle coupled with an nickel
ion.
[0080] According to the present invention, up to 16 kinds of target
proteins may be synthesized and produced within 6 hours by
combining the method for purification proteins using the magnetic
particle with the method for expressing the cell-free protein and
applying the combined methods to the automatic biological material
purification apparatus including a heating part and a magnetic
field applying part.
Advantageous Effects
[0081] The method for producing proteins according to the present
invention uses the automatic biological material purification
apparatus including: a well plate kit; a heating part; and a
magnetic field applying part, such that a plurality of target
proteins may be more quickly and simply obtained as compared to
target proteins obtained by using the existing method for
expressing/purifying proteins through conventional cell culture,
and a reproducible synthesis efficiency on the same proteins may be
obtained due to no deviation between reaction wells.
DESCRIPTION OF DRAWINGS
[0082] FIG. 1 shows a state in which a base plate of an automatic
biological material purification apparatus including a heating part
and a magnetic applying part according to the present invention
inserts a casing;
[0083] FIG. 2 schematically shows a pipette block of the automatic
biological material purification apparatus including a heating part
and a magnetic applying part according to the present
invention;
[0084] FIG. 3 is a cross-sectional view of a main part of the
pipette block of the automatic biological material purification
apparatus including a heating part and a magnetic applying part
according to the present invention;
[0085] FIG. 4 is a perspective view in which a casing is partially
removed from the automatic biological material purification
apparatus including a heating part and a magnetic applying part
according to the present invention;
[0086] FIG. 5 shows a state in which a base plate of the automatic
biological material purification apparatus including a heating part
and a magnetic applying part according to the present invention is
used;
[0087] FIG. 6 is a perspective view of a magnetic mounting part and
a lifting part of the automatic biological material purification
apparatus including a heating part and a magnetic applying part
according to the present invention;
[0088] FIG. 7 shows a manufacturing diagram of a template PCR
product for producing proteins using the automatic biological
material purification apparatus including a heating part and a
magnetic applying part according to the present invention;
[0089] FIG. 8 is a diagram of a method for producing proteins using
the automatic biological material purification apparatus including
a heating part and a magnetic applying part according to the
present invention;
[0090] FIG. 9 is a diagram of an experiment using an automatic
biological material purification apparatus including a heating part
and a magnetic applying part according to the present
invention;
[0091] FIG. 10 shows a first multi well plate kit (A) and a second
multi well plate kit (B) used in the automatic biological material
purification apparatus including a heating part and a magnetic
applying part according to the present invention;
[0092] (Column G: Well region having a cell-free protein expression
solution, Column H: Well region having a DEPC dilution water,
Column L: Well region having a template for cell-free protein
synthesis, Column A to D: Well region having a magnetic particle
reaction solution and a washing solution for coupling a target
protein with the magnetic particles, Column E: Well region having a
target protein elution, and Column F: Well region having magnetic
particles for attaching the target protein)
[0093] FIG. 11 shows a result of a SDS-PAGE gel of target proteins
(GFPs) produced by using a method for producing proteins according
to the present invention; and
[0094] (M: AccuLadder.TM. Protein Size Marker (Low) of Bioneer
Corporation, E: expressed GFP sample, P: purified GFP sample,
1.about.16: GFP synthesis protein reacted in each unit well)
[0095] FIG. 12 shows a result of SDS-PAGE gel of target proteins
for various template DNAs produced by using a method for producing
proteins according to the present invention.
DETAILED DESCRIPTION OF MAIN ELEMENTS
[0096] 100: pipette BLOCK 110: SYRINGE PIN HOLDER
[0097] 112: INFORMATION GUIDE 120: SYRINGE PIN
[0098] 130: SYRINGE PIN GUIDE BLOCK 131: SYRINGE PIN GUIDE HOLE
[0099] 133: pipette MOUNTING PART 133-1: COMMUNICATING HOLE
[0100] 133-2: ADHESION RING 134: pipette MOUNTING PART
[0101] 134-1: COMMUNICATING HOLE 134-2: ADHESION RING
[0102] 141: pipette 142: pipette
[0103] 150: SYRINGE PIN GUIDE BLOCK SUPPORTING PLATE 152:
UP-AND-DOWN MOVING NUT
[0104] 160: GUIDE ROD
[0105] 171: SYRINGE PIN CONTROL MOTOR SUPPORTING PLATE
[0106] 172: SYRINGE PIN CONTROL MOTOR
[0107] 173: SYRINGE PIN CONTROL SCREW
[0108] 181: UPPER DESORPTION PLATE 182: DESORPTION PLATE
[0109] 183: CONNECTION ROD 184: PROTRUSION ROD
[0110] 185: SPRING 200: FIXED MAIN BODY
[0111] 231: UP-AND-DOWN MOVING MOTOR
[0112] 232: UP-AND-DOWN MOVING BELT
[0113] 233: UP-AND-DOWN MOVING SCREW
[0114] 241: BACK-AND-FORTH MOVING SLIDER
[0115] 300: CASING 310: BACK-AND-FORTH MOVING SUPPORT ROD
[0116] 311: BACK-AND-FORTH GUIDER 350: DOOR
[0117] 320: BACK-AND-FORTH MOVING MOTOR
[0118] 330: BACK-AND-FORTH MOVING BELT
[0119] 340: ULTRAVIOLET LAMP 360: TOUCH SCREEN
[0120] 400: BASE PLATE 401: DOORNOB
[0121] 420': MULTI WELL PLATE 420: MULTI WELL PLATE
[0122] 430: pipette RACK 440: PROTEIN STORAGE TUBE RACK
[0123] 450: WASTE tray
[0124] 442-1: CELL DISRUPTED LIQUID STORAGE TUBE
[0125] 442-3: PROTEIN STORAGE TUBE
[0126] 700: MAGNETIC FIELD APPLYING PART
[0127] 710: MAGNET MOUNTING PART 711: MAGNET
[0128] 713: UNIT WELL INSERTING GROOVE
[0129] 720: MAGNET MOUNTING PART SUPPORT
[0130] 730: GUIDE ROD 740: GUIDE BLOCK
[0131] 750: TENSION SPRING 760: LIFTING PART
[0132] 761: LIFTING MOTOR 762: FIRST LIFTING SHAFT
[0133] 763: LIFTING CAM 764: SECOND LIFTING SHAFT
[0134] 780: HEIGHT SENSOR 781: SENSING PART
[0135] 782: SENSING TARGET PART 810: HEATING PART
[0136] 812: HEATING PART FIXING PLATE
BEST MODE
[0137] Hereinafter, although the present invention will be
described in detail with reference to the following Examples, which
has been disclosed for illustrative purposes, it will be apparent
to those skilled in the art that modifications and variations can
be made without being limited by the following Examples. Materials
and methods used in Examples of the present invention are as
follows.
EXAMPLE 1
Preparation of Template DNA
[0138] (1) Preparation of Plasmid DNA
[0139] A template DNA-plasmid DNA was prepared for protein
synthesis. Each gene was synthesized by a method for gene synthesis
(NBiochem. Biophys. Res. Commun. 1998, 248, 200-203), followed by
treatment with a restriction enzyme, and cloning into an expression
vector for E. coli.
[0140] As the expression vector for E. coli, pBIVT (Bioneer
Corporation, Korea), pIVEX (Roche, Germany), pET (Novagen,
Germany), pK7, pQE, and the like, may be used, but the present
invention is not limited thereto.
[0141] In addition, the expression vector for E. coli, an affinity
tag should be included in the expression vector. In the present
Example, the expression vector including a histidine tag was used,
and but the present invention is not limited thereto and thus, the
expression vector may include another tag.
[0142] In the present Example, CalmL3, RNaseH, DUSP3, CAT, AcGFP,
EF-Ts, VF, Poly A polymerase, MMLV RTase, BM3, gene were used.
[0143] More specifically, for the treatment with the restriction
enzyme on a gene synthesis material, 1 .mu.l of BamHI (Bioneer
Corporation, Korea), 1 .mu.l of NotI (Bioneer Corporation, Korea),
2 .mu.l of 10.times. AccuCut.TM. buffer (Bioneer Corporation,
Korea), 10 .mu.l of gene synthesis material and 6 .mu.l of sterile
distilled water were added to each tube, mixed together, and
disposed at a constant temperature of 37.degree. C. for three
hours. For the treatment with the restriction enzyme on an
expression vector for E. coli, 1 .mu.l of BamHI (Bioneer
Corporation, Korea), 1 .mu.l of NotI (Bioneer Corporation, Korea),
2 .mu.l of 10.times. AccuCut.TM. buffer (Bioneer Corporation,
Korea), 10 .mu.l of expression vector for E. coli and 6 .mu.l of
sterile distilled water were added to each tube, mixed together,
and disposed at a constant temperature of 37.degree. C. for three
hours. DNA was extracted from each reactant treated with each
restriction enzyme using Accuprep Gel Extraction kit (Bioneer
Corporation, Korea).
[0144] 5 .mu.l of 2.times. rapid ligation buffer (Promega
Corporation, US), 1 .mu.l of T4 DNA ligase (Promega Corporation,
US), 3 .mu.l of restriction enzyme treatment gene synthesis
material, 1 .mu.l of restriction enzyme treatment vector were added
to a tube, mixed together, and disposed at a constant temperature
of 16.degree. C. for one hour. Then, 10 .mu.l of the reaction
solution disposed at constant temperature was put into 100 .mu.l of
E. coli competent cell, the mixture was placed on ice for 30
minutes and cultured at 42.degree. C. for 90 seconds, and then
placed again on ice for 5 minutes. The reactant was inoculated into
a LB plate containing kanamycin and cultured at 37.degree. C. for
16 hours.
[0145] After white colony was taken and cultured in 10 .rho.l of LB
liquid medium for 16 hours, followed by centrifugation, the
thus-obtained supernatant was removed and plasmid DNA was extracted
from pellet using AccuPrep plasmid DNA prep kit (Bioneer
Corporation, Korea). It was confirmed by sequencing whether or not
the extracted DNA was gene synthesized by each gene synthesis
method, and as DNA to be used for protein synthesis, corresponding
colony was cultured to secure plasmid DNA by the same method.
Concentration and purity of the plasmid DNA were measured by UV
spectrometer (Shimazu Corporation, Japan) and it was confirmed that
the plasmid DNA had a purity of 1.8 to 2.0.
[0146] (2) Preparation of PCR Product
[0147] A template DNA-PCR product was prepared for protein
synthesis. A `kit for PCR` using two-step PCR principle has been
developed for preparation of PCR product, and a method for
preparing PCR product is shown in FIG. 7 and kit components are
shown in the following Table 1.
TABLE-US-00001 TABLE 1 Kit for PCR AccuPower .RTM. PCR Premix N
terminus upstream cassette N terminus downstream cassette C
terminus upstream cassette C terminus downstream cassette 2.sup.nd
Forward primer 2.sup.nd Reverse primer
[0148] More specifically, a target gene was firstly capable of
being amplified, a primer set having overlapping sequences at 5'
and 3' terminals was fabricated, and a first PCR reaction was
performed with a sample having target gene (genomic DNA, T vector,
and the like) as a template. In order to perform PCR reaction,
AccuPower PCR Premix provided in a kit was used and the reaction
under reaction conditions including denaturalization at 94.degree.
C. for 5 minutes and amplification at 94.degree. C. for 30 seconds,
at 58.degree. C. for 30 seconds and at 72.degree. C. for 1 minute
as one cycle was performed thirty times, and then polymerization
was finally performed at 72.degree. C. for 5 minutes. Then, a
primary PCR reactant was refined using AccuPrep PCR refinement kit
(Bioneer Corporation, Korea) or AccuPrep Gel Extraction kit
(Bioneer Corporation, Korea).
[0149] A secondary overlapping PCR was performed using the primary
PCR reactant as a template to finally synthesize a PCR product for
protein synthesis. In order to perform the PCR reaction, a cassette
set and a primer set provided in a kit of Table 1 above and the
primary PCR reactant were added and the reaction under the reaction
conditions including denaturalization at 94.degree. C. for 5
minutes and amplification at 94.degree. C. for 30 seconds, at
58.degree. C. for 30 seconds and at 72.degree. C. for 1 minute as
one cycle was performed thirty times, and then polymerization was
finally performed at 72.degree. C. for 5 minutes. A secondary PCR
reactant was refined using AccuPrep Gel Extraction kit (Bioneer
Corporation, Korea).
[0150] The upstream cassette and downstream cassette as provided
above are oligonucleotides encoding an affinity tag coupled to the
target protein, and more preferably, they include a histidine
tag.
EXAMPLE 2
Preparation of Cell Disrupted Liquid for Protein Expression
[0151] (1) Cell Culture
[0152] First, E. coli [BL21(DE3) (Novagen Corporation, US)] was
cultured in 350 l fermenter (2.times. YT medium) at 37.degree. C.
Then, at absorbance (OD 600) of 0.5, 1 mM IPTG was added to express
T7 RNA polymerase, and at absorbance (OD 600) 3.0 to 6.0, culturing
was terminated and cells were recovered by centrifugation and
stored at -50.degree. C.
[0153] (2) Preparation of Cell Disrupted liquid
[0154] The recovered E. coli 100 g was added to 500 ml of washing
solution [10 mM Tris(oAc) pH 8.2, 14 mM Mg(oAc).sub.2, 60 mM
K(OAc), 1 mM DTT (dithiothreitol), and 0.05% (v/v)
2-mercaptoethanol (2-ME)] and well-washed, followed by
centrifugation (3,000 RPM, 30 minutes), and the above-described
procedures were repeated three times. After washing and measuring
E. coli mass, a buffer solution [2-ME was removed from the washing
solution] having a volume 1.1 times larger than the mass was added,
E. coli was uniformly mixed, and then cells were disrupted under a
constant pressure (160 to 280 MPa) using a pressure cell
homogenizer (Stansted Fluid Power).
[0155] The cell disrupted liquid was separated by high speed
centrifugation (16,000 RPM, 30 minutes, 4.degree. C.) to recover
supernatant, and a pre-culture solution [293.3 mM Tris(OAc) pH 8.2,
2 mM Mg(OAc).sub.2, 10.4 mM ATP, 200 mM creatine phosphate, 4.4 mM
DTT, 0.04 mM amino acids, and 26.7 g/ml creatine kinase] was added
to the cell disrupted liquid, wherein 3 ml of the pre-culture
solution was used per 10 ml of the cell disrupted liquid, followed
by culturing at 37.degree. C. for 90 minutes, thereby preparing a
pre-cultured solution. In addition, after the pre-cultured solution
was put into a dialysis tube (10 kDa, Dialysis Tubing, Sigma
Corporation, US), and dialyzed four times in buffer solution having
20 times volume at 4.degree. C. for 45 minutes, thereby removing
foreign materials after the pre-culture, and a solution in the
dialysis tube was subjected to centrifugation (11,000 RPM, 20
minutes, 4.degree. C.), and cell disrupted liquid for protein
synthesis was prepared. The cell disrupted liquid was stored at
-70.degree. before fabricating a multi well plate kit.
EXAMPLE 3
Preparation of Cell-Free Protein Expression Solution
[0156] A protein expression solution required for cell-free protein
synthesis was prepared.
[0157] More specifically, the cell-free protein expression solution
[114 mM Hepes-KOH (pH 8.2), 2.4 mM ATP, each of 1.7 mM CTP, GTP and
UTP, 2 mM DTT, 90 mM K (Glu), 80 mM NH4(OAc), 12 mM Mg(OAc), 68
g/ml folinic acid (L-5-formyl-5,6,7,8-tetrahydrofolic acid), each
of 1.5 mM 20 amino acids, 2% PEG 8000, and 67 mM creatine
phosphate] was prepared. The expression solution was stored at
-20.degree. C. before fabricating a multi well plate kit.
EXAMPLE 4
Preparation of Magnetic Particle Reaction Solution and Washing
Solution
[0158] A magnetic particle used for protein purification is a
magnetic particle obtained by coating Fe particles having magnetism
with silica and complexing an end thereof with nickel (Ni). The
magnetic particle is commercially available from Sigma Corporation,
Promega Corporation, Qiagen Corporation, and the like. The magnetic
bead has advantages in that protein is capable of easily refined
using magnetic properties, thereby making it possible to be used in
various fields.
[0159] The magnetic particle reaction solution for coupling the
target protein with the magnetic particles required for extraction
of the target protein and the washing solution were prepared. More
specifically, the magnetic particle reaction solution and the
washing solution [50 mM HEPES-KOH (pH7.5), 300 mM NaCl, 10 mM
Imidazole, 5 mM 2-mercaptoethanol (2-ME) and 10% (v/v) glycerol]
were prepared. The solutions was stored at 4.degree. C. before
fabricating a multi well plate kit.
EXAMPLE 5
Preparation of Protein Eluting Solution
[0160] A protein eluting solution required for purification of the
target protein was prepared.
[0161] More specifically, a protein eluting solution [50 mM
HEPES-KOH (pH7.5), 300 mM NaCl, 1 M Imidazole, 5 mM
2-mercaptoethanol (2-ME), 10% (v/v) glycerol] were prepared. The
solutions was stored at 4.degree. C. before fabricating a multi
well plate kit.
EXAMPLE 6
Manufacture of Multi Well Plate Kit
[0162] (1) Division and Storage of Cell Disrupted Liquid
[0163] The stored cell disrupted liquid prepared by Example 2 above
was completely melted on an ice. The completely melted cell
disrupted liquid was divided into 8-strip tube for 200 .mu.l each,
and stored at -70.degree. C.
[0164] (2) Manufacture of Multi Well Plate Kit
[0165] Multi well plate kits were manufactured using solutions
prepared by Examples 3 to 5 above.
[0166] First, referring to B of FIG. 10, a second multi well plate
kit 420' was manufactured. More specifically, after the cell-free
protein expression solution prepared by Example 3 above was
completely melted on an ice, a protein expression solution of 350
.mu.l was divided into each portion of column G of the multi well
plate, a DEPC distilled water (Bioneer Corporation, Korea) of 200
.mu.l was divided into each portion of column H thereof shown in B
of FIG. 10, and then, an upper portion of the multi well plate was
sealed with a film and stored at -20.degree. C.
[0167] Then, referring to A of FIG. 10, a first multi well plate
kit 420 was manufactured . More specifically, the magnetic particle
reaction solution and the washing solution prepared in Example 4
above of 1.2 ml were divided into each portion of columns A to D of
the multi well plate 1 shown in A of FIG. 10, and the protein
eluting solution of 250 .mu.l was divided into each portion of
column E. In addition, a solution containing magnetic particles
coupled with an nickel ion (Bioneer Corporation, Korea) of 500
.mu.l was divided into each portion of column F, and then, an upper
portion of the multi well plate was sealed with a film and stored
at 4.degree. C.
EXAMPLE 7
Method for Expressing/Purifying Protein Using Automatic Biological
Material purification Apparatus Including Heating Part and Magnetic
Field Applying Part
[0168] (1) Preparation for Protein Synthesis
[0169] As an automatic biological material purification apparatus
including a heating part and a magnetic field applying part used in
the present invention, an apparatus disclosed in Korean Patent No.
KR 10-1025135, Korean Patent Laid-Open Publication No.
10-2011-0081718, and the like, may be used or ExiProgen.TM.
(Bioneer Corporation, Korea) may be used, but the present invention
is not limited thereto. In the present experiment, ExiProgen.TM.
(Bioneer Corporation, Korea) was used.
[0170] A method for expression/purifying protein using an automatic
biological material purification apparatus including a heating part
and a magnetic field applying part was performed as shown in FIG.
9. More specifically, the tube having the divided cell disrupted
liquid prepared by step (1) of Example 6 and the multi well plate
kit were taken out from fridge to be completely melted at room
temperature. Then, first and second multi well plates were punched
using 6 Hole Puncher which is provided with ExiProgen.TM. (Bioneer
Corporation, Korea), the automatic biological material purification
apparatus including a heating part and a magnetic field applying
part.
[0171] The template DNA prepared by Example 1 was added to column L
of the second multi well plate 420'. Then, the cell disrupted
liquid and the multi well plates were mounted on corresponding
positions of ExiProgen.TM. (Bioneer Corporation, Korea). After
elution tube to be filled with the protein purification solution
and a filter tip were put into corresponding racks and mounted on
positions of the corresponding racks, a set up tray was pushed
therein, and a door of the apparatus was closed. ExiProgen.TM.
(Bioneer Corporation, Korea) was turned on and protocol 902 was
practiced.
[0172] Here, an amount of the template DNA to be added may vary
depending on kinds and sizes of the template DNA, preferably, in
the case of plasmid DNA, 1 to 10 ug was used, and in the case of
PCR product, 500 ng to 2 ug was used.
[0173] (2) Process for Expressing/Purifying Protein Using Automatic
Biological Material purification Apparatus Including Heating Part
and Magnetic Field Applying Part
[0174] FIG. 8 is a diagram of a method for producing proteins
simultaneously performing expression and purification of target
protein using an automatic biological material purification
apparatus including a heating part and a magnetic applying
part.
[0175] Referring to FIGS. 5 and 8, an injecting step S10 of target
DNA as a template for cell-free protein synthesis into the specific
unit well was provided. Referring to B of FIG. 10, DNA was added to
a unit well L of the multi well plate 420' in the injecting step
S10 of target DNA.
[0176] Referring to FIGS. 5 and 8, a first mixing step S20 mixing
by injecting a DEPC distilled water into the specific unit well was
provided. In the mixing step by injecting the DEPC distilled water
into the specific unit well, the DEPC distilled water injected into
a unit well H of the multi well plate 420' was injected into the
specific unit well L using pipettes 141 and 142 (see FIG. 3).
Accordingly, the DEPC distilled water was mixed with the target DNA
injected into the specific unit well L.
[0177] Referring to FIGS. 5 and 8, a second mixing step S30 mixing
by injecting a cell free protein expression solution into the
specific unit well was provided. In the second mixing step S30, the
cell free protein expression solution injected into a unit well G
of the multi well plate 420' was injected into the specific unit
well L using pipettes 141 and 142. Accordingly, the cell free
protein expression solution was mixed with the target DNA solution
in the specific unit well L.
[0178] Referring to FIGS. 5 and 8, a mixture preparing step S40
mixing by injecting a cell disrupted liquid into the specific unit
well was provided. In the mixture preparing step S40, the cell
disrupted liquid injected into a tube 442-1 (see FIG. 5) injected
with the cell disrupted liquid was injected into the specific unit
well L using the pipettes 141 and 142. Accordingly, the cell
disrupted liquid was mixed with the mixture of the target DNA and
the cell free protein expression solution in the specific unit well
L, thereby preparing a protein synthesis reaction solution.
[0179] Referring to FIGS. 5 and 8, a first heating step S50 was
provided. In the heating step S50, a state in which the lower
portion of the specific unit well L of the multi well plate kit
420' inserted a unit well inserting groove 713 (see FIG. 6) by
lifting up the magnet mounting part 710 (see FIG. 6) was provided.
Then, expression of protein in the prepared protein synthesis
reaction solution is promoted by heating a lower portion of the
specific unit well L using the heating part 720 (see FIG. 6). In
the heating step S50, the reaction is activated due to enzymes in
the mixture to achieve RNA synthesis from target DNA and protein
expression. The lower portion of the specific unit well L may be
heated at 30.degree. C. for 3 hours by the heating part.
[0180] Referring to FIGS. 5 and 8, a first protein expression
injecting step S60 into the specific unit well was provided. In the
first protein expression injecting step S60, the protein expressed
mixture from the specific unit well L after the first heating step
S50 was injected into a unit well J of the multi well plate 420'
using the pipettes 141 and 142.
[0181] Referring to FIGS. 5 and 8, a third mixing step S70 mixing
the magnetic particle reaction solution and the magnetic particles
is provided. In the third mixing step S70, the magnetic particle
reaction solution injected into a unit well A of the multi well
plate 420 was injected into the unit well F of the multi well plate
420 using the pipettes 141 and 142 to be mixed with the magnetic
particles. Accordingly, a surface of the magnetic particle was
equilibrated by the magnetic particle reaction solution.
[0182] Referring to FIGS. 5 and 8, the injecting step of the
magnetic particle into the specific unit well, that is, the second
injecting step S80 of the mixture mixed with the magnetic particle
reaction solution into the specific unit well was provided. In the
injecting step S80, the mixture mixed with the magnetic particle
reaction solution of the specific unit well F was injected into the
specific unit well L using the pipettes 141 and 142 in a state in
which the magnet mounting part 710 is lifted down.
[0183] Referring to FIGS. 5 and 8, a first magnetic field applying
step S90 was provided. In the first magnetic field applying step
S90, the lower portion of the specific unit well L inserted the
unit well inserting groove 713 by lifting up the magnet mounting
part 710. Accordingly, a magnetic field was applied from the magnet
711 (see FIG. 6) mounted on the magnet mounting part to the lower
portion of the specific unit well L.
[0184] Referring to FIGS. 5 and 8, a first removing step S100 was
provided. The first removing step S100 was performed in a state in
which a magnetic field is applied to the lower portion of the
specific unit well by the first magnetic field applying step S90.
More specifically, in the first removing step S100, a state in
which the magnetic particles of the magnetic particle reaction
solution in the mixture mixed with the magnetic particle reaction
solution were attached to an inner wall of the lower portion of the
specific unit well L was maintained.
[0185] In addition, in the first removing step S100, the reaction
solution except for the magnetic particles in the mixture mixed
with the magnetic particle reaction solution was removed using the
pipettes 141 and 142. The reaction solution removed from the first
removing step S100 may be discharged to a waste tray 450 (see FIG.
5). The first removing step S100 was performed, such that the
magnetic particles were left in the specific unit well L.
[0186] In addition, the injecting step S80 of the reaction solution
mixed with the magnetic particles into the specific unit well, the
first magnetic field applying step S90, and the first removing step
S100 may be repeated once. Here, in the injecting step S80 of the
reaction solution mixed with the magnetic particles into the
specific unit well, the magnetic particle reaction solution
injected into the unit well A of the multi well plate 420 was
injected into the specific unit well L. Then, the first magnetic
field applying step S90 and the first removing step S100 were
performed in the same scheme.
[0187] Referring to FIGS. 5 and 8, a second protein expression
injecting step S110 into the specific unit well was provided. In
the second protein expression injecting step S110, the protein
expression of the specific unit well J was injected into the
specific unit well L using the pipettes 141 and 142.
[0188] Referring to FIGS. 5 and 8, reacting step S120 mixing the
second protein expression with the magnetic particles was provided.
In the reacting step S120, the second protein expression injected
into the specific unit well L was mixed with the magnetic particles
using the pipettes 141 and 142. Accordingly, a surface of the
magnetic particle was coupled with the protein expression.
[0189] Referring to FIGS. 5 and 8, a second magnetic field applying
step S130 was provided. In the second magnetic field applying step
S130, the lower portion of the specific unit well L inserted the
unit well inserting groove 713 by lifting up the magnet mounting
part 710. Accordingly, a magnetic field was applied from the magnet
mounted on the magnet mounting part 710 to the lower portion of the
specific unit well L.
[0190] Referring to FIGS. 5 and 8, a second removing step S140 was
provided. The second removing step S140 was performed in a state in
which a magnetic field is applied to the lower portion of the
specific unit well L by the second magnetic field applying step
S130. Therefore, in the second removing step S130, a state in which
magnetic particles and protein coupled with the magnetic particles
in the magnetic particle protein expression were attached to an
inner wall of the lower portion of the specific unit well L by a
magnetic field was maintained.
[0191] In addition, in the second removing step S140, a mixture
except for magnetic particles and proteins coupled with the
magnetic particles in the magnetic particle protein expression was
removed using the pipettes 141 and 142. The mixture removed from
the second removing step S140 may be discharged to a unit well K of
the multi well plate 420'. The second removing step S140 was
performed, such that the magnetic particles and the proteins
coupled with the magnetic particles were left in the specific unit
well L.
[0192] Referring to FIGS. 5 and 8, a washing step S150 separating
impurities except for target protein from the magnetic particles by
injecting a washing solution into the specific unit well and
washing the magnetic particles was provided. In the washing step
S150, the washing solution injected into the unit well B of the
multi well plate 420 was injected into the specific unit well using
the pipettes 141 and 142 in a state in which the magnet mounting
part 710 is lifted down and mixed with together to thereby separate
impurities except for target protein from the magnetic
particles.
[0193] Referring to FIGS. 5 and 8, a third magnetic field applying
step S160 was provided. In the third magnetic field applying step
S160, the lower portion of the specific unit well L inserted the
unit well inserting groove by lifting up the magnet mounting part
710. Accordingly, a magnetic field was applied from the magnet 711
mounted on the magnet mounting part 710 to the lower portion of the
specific unit well L.
[0194] Referring to FIGS. 5 and 8, a third removing step S170 was
provided. The third removing step S170 was performed in a state in
which a magnetic field is applied to the lower portion of the
specific unit well L by the third magnetic field applying step
S160. Therefore, in the third removing step S170, a state in which
magnetic particles having the target proteins attached thereto in
the mixture mixed with the washing solution were attached to an
inner wall of the lower portion of the specific unit well L by a
magnetic field was maintained.
[0195] In addition, in the third removing step S170, a mixture
except for magnetic particles having the target proteins attached
thereto in the mixture mixed with the washing solution was removed
using the pipettes 141 and 142. The mixture removed from the third
removing step S170 may be discharged to a unit well I of the multi
well plate 420'. The third removing step S170 was performed, such
that the magnetic particles having the target proteins attached
thereto were left in the specific unit well L.
[0196] Washing step S150 separating impurities except for target
protein from the magnetic particles by injecting a washing solution
into the specific unit well and washing the magnetic particles, the
third magnetic applying unit S160, and the third removing step S170
may be sequentially repeated several times and the mixture removed
from the third step S170 is discharged to the waste tray 450.
[0197] Referring to FIGS. 5 and 8, a target protein separating step
S180 purifying the target protein by injecting a protein eluting
solution into the specific unit well was provided. In the target
protein separating step S180, the protein eluting solution injected
into the unit well E of the multi well plate 420 was injected into
the specific unit well L using the pipettes 141 and 142 in a state
in which the magnet mounting part 710 is lifted down and mixed with
together to thereby separate the target protein from the magnetic
particles.
[0198] Referring to FIGS. 5 and 8, a fourth magnetic field applying
step S190 was provided. The fourth magnetic field applying step
S190 was performed after the target protein separating step S180
purifying the target protein by injecting the protein eluting
solution into the specific unit well and then a predetermined time
is passed. In the fourth magnetic field applying step S190, the
lower portion of the specific unit well L inserted the unit well
inserting groove 713 by lifting up the magnet mounting part 710.
Accordingly, a magnetic field was applied from the magnet mounted
on the magnet mounting part 710 to the lower portion of the
specific unit well L.
[0199] Referring to FIGS. 5 and 8, a target protein containing
solution obtaining step S200 was provided. The target protein
containing solution obtaining step S200 was performed in a state in
which a magnetic field is applied to the lower portion of the
specific unit well L by the fourth magnetic field applying step
S190. Therefore, in the target protein containing solution
obtaining step S200, a state in which the magnetic particles in the
protein eluting solution containing the proteins separated from the
magnetic particles were attached to an inner wall of the lower
portion of the specific unit well L was maintained.
[0200] In the target protein containing solution obtaining step
S200, the target protein containing solution which is a mixture
except for the magnetic particles in the protein eluting solution
containing the proteins separated from the magnetic particles was
injected and stored into a protein storage tube 442-3 mounted on
the base plate 400 using the pipettes 141 and 142.
[0201] (3) Experimental Results Using Protein Synthesis Kit
[0202] Results obtained by simultaneously synthesizing GFP in 16
reaction wells using the kit according to the present invention and
ExiProgen.TM. (Bioneer Corporation, Korea) by Example 7 were shown
in FIG. 11. More specifically, the results were confirmed in 10 to
12% SDS-PAGE gel, and it could be appreciated that a reproducible
synthesis efficiency on the same proteins may be obtained due to no
deviation between reaction wells.
[0203] In addition, results obtained by synthesizing each different
protein using templates fabricated in various forms were shown in
FIG. 12.
* * * * *